Apoprotein (apo) E is a well-characterized protein whose acknowledged function is to mediate the transport and uptake of cholesterol-rich plasma lipoproteins. Incongruous with this role in cholesterol homeostasis is the repeated observation that apo E is also synthesized in multiple extrahepatic sites including ovarian granulosa cells. While investigating the lipoprotein requirements of ovary androgen producing cells, I discovered that apo E is a specific inhibitor of androgen synthesis. In the presence of apo E, cultured LH-stimulated theca/interstitial (T/I) cells make progesterone, but cannot convert it to androgen. Furthermore, I have recently identified a synthetic peptide analogue of apo E that is capable of mimicking intact apo E. Granulosa cells and theca cells are dependent on one another to coordinate the production of estrogen that is critical in controlling follicular development. To test the hypothesis that granulosa cell apo E is an intraovarian regulator of T/I cell androgen production, five Specific Aims will be accomplished. The first is to demonstrate that granulosa cell-derived apo E inhibits T/I cell androgen production. Apo E will be isolated from granulosa cell culture supernatants and tested for biologic activity. The second Specific Aim is to characterize the changes in ovarian apo E content, apo E synthesis, and apo E binding during follicular development. Using immunohistochemical and autoradiographic techniques, apo E and its binding sites will be identified in follicles at different stages of development. In addition, cells expressing apo E mRNA will be identified by In situ hybridization. The third Specific Aim is to characterize the interaction of apo E with cultured T/I cells and to identify the molecular nature of the cellular binding sites. Using both granulosa cell-derived apo E and a synthetic apo E peptide analogue, binding to T/I cells will be characterized. The fourth Specific Aim is to define the structural features of apo E that are responsible for its binding to and biologic effect on T/I cells. Chemical modifications and amino acid substitutions will be made in the apo E synthetic peptide to define the structural requirements for both binding and function. The fifth Specific Aim is to identify the site of the inhibitory activity of apo E pre- and post-cAMP mediation of theca/interstitial cell differentiation. Based on the information obtained from this investigation, it will be possible to assess the physiologic role of granulosa cell apo E in ovarian function.